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Two clonal rat rhabdomyosarcoma cell lines BA-Han-1B and BA-Han-1C with different capacities for myogenic differentiation have been examined for the expression of muscle regulatory basic helix-loop-helix (bHLH) proteins of the MyoD family. Whereas cells of the BA-Han-1C subpopulation constitutively expressed MyoD1 and could be induced to differentiate with retinoic acid (RA), BA-Han-1B cells did not express any of the myogenic control factors and appeared to be largely differentiation-defective. Upon induction with RA, BA-Han-1C cells expressed also myogenin, in contrast to BA-Han-1B cells which never activated any of the genes encoding muscle bHLH factors. The onset of myogenin transcription in BA-Han-1C cells required de novo protein synthesis and DNA replication suggesting that RA probably did not act directly on the myogenin gene. Although MyoD1 was expressed in proliferating BA-Han-1C myoblasts, muscle-specific reporter genes were not activated indicating that MyoD was biologically inactive. However, transfections with plasmid expressing additional MyoD1 protein resulted in the transactivation of muscle genes even in the absence of RA. mRNA encoding the negative regulatory HLH protein Id was expressed in proliferating BA-Han-1C cells and disappeared later after RA induction which suggested that it may be involved in the regulation of MyoD1 activity. The myogenic differentiation of malignant rhabdomyosarcoma cells strictly correlated with the activation of the myogenin gene. In fact, stable transfections of BA-Han-1C cells with myogenin expressing plasmids resulted in spontaneous differentiation. Together, our results suggest that the transformed and undifferentiated phenotype of BA-Han-1C rhabdomyosarcoma cells is associated with the inactivation of the myogenic factor MyoD1 as well as lack of myogenin expression. RA alleviates the inhibition of myogenic differentiation, probably by activating MyoD protein and myogenin gene transcription. BA-Han-1B cells did not respond to RA and the differentiated phenotype could not be restored by overexpression of MyoD1 or myogenin.

We have isolated and sequenced the gene encoding the human embryonic/atrial myosin alkali light chain isoform ( MLC-1 emb A ). The gene is split into seven exon by six introns; the last exon, as in all MLC isoform genes sequenced to date, is completely 3′ untranslated sequence. Comparison of the MLC-1 emb A isoform gene with the other MLC-1 genes showed that the exonintron arrangement of the human MLC-1 emb A isoform gene is analogous to that of the other MLC-1 type isoform genes. We have also mapped the human MLC-1 emb A isoform gene to the long arm of chromosome 17; the corresponding mouse gene has been mapped to chromosome 11. This gene, together with a number of others such as the collagen(I) α1, galactokinase, and thymidine kinase genes, is part of the largest syntenic group between mouse and man.

MyoD is a member of a family of myogenic transcription factors with similar properties [1] including myogenin [2–4], Myf-5 [5], and Myf-6, also called MRF4 or herculin [6–8]. Transfection of the myogenic HLH genes into a wide range of cultured cells can induce the skeletal muscle differentiation program and activate muscle-specific genes. No substantial difference between the myogenic HLH genes has been revealed by these assays. The myogenic transcription factor genes are expressed solely in skeletal muscle, and thus, their role appears to be in determining the identity of the skeletal myocyte lineage [9–12].

The Myf-5 gene, a member of the myogenic basic HLH factor family, has been inactivated in mice after homologous recombination in ES cells. Mice lacking Myf-5 were unable to breathe and died immediately after birth, owing to the absence of the major distal part of the ribs. Other skeletal abnormalities, except for complete ossification of the sternum, were not apparent. Histological examination of skeletal muscle from newborn mice revealed no morphological abnormalities. Northern blot analysis demonstrated normal levels of muscle-specific mRNAs including MyoD, myogenin, and Myf-6. However, the appearance of myotomal cells in early somites was delayed by several days. These results suggest that while Myf-5 plays a crucial role in the formation of lateral sclerotome derivatives, Myf-5 is dispensable for the development of skeletal muscle, perhaps because other members of the myogenic HLH family substitute for Myf-5 activity.

Myf-5 is a member of a family of muscle-specific transcription factors that activate myogenesis in 10T1/2 fibroblasts. Here we report the analysis of Myf-5 structural domains that are responsible for its biological activity. Site-directed mutagenesis revealed that two clusters of basic amino acids within a conserved basic region and two amphipathic helices within the adjacent HLH domain are essential for sequence-specific DNA binding and hetero-oligomerization, respectively. Transcriptional activation by Myf-5 requires two additional domains located in the amino- and carboxyl-termini. The two domains apparently co-operate since deletion of either one results in inactivation. Chimeric proteins between DNA binding domain of the yeast transcription factor GAL4 and the separate Myf-5 transactivator domains exhibit activity that is enhanced when both regions are combined. Dimerization of Myf-5 with the ubiquitously expressed bHLH protein E12 not only increases the affinity for DNA but also stimulates transactivation independently of DNA binding. The Myf-5 transactivator domains are dependent for activity on a specific amino acid sequence motif within the basic region when Myf-5 activity is mediated through the E-box DNA recognition sequence but not when DNA binding occurs through the GAL4 DNA binding domain. This demonstrates that muscle-specific transactivation by Myf-5 requires the collaboration of two activation domains and the DNA binding region in addition to sequence-specific DNA binding. Transcriptional activation and interaction with DNA are executed by separable domains; however, transactivation is influenced by the basic region in a manner distinguishable from DNA binding.

The spatial and temporal expression pattern of the muscle regulatory gene Myf-6 (MRF4/herculin) has been investigated by in situ hybridization during embryonic and fetal mouse development. Here, we report that the Myf-6 gene shows a biphasic pattern of expression. Myf-6 transcripts are first detected in the most rostral somites of the mouse embryo at 9 d of gestation and accumulate progressively in myotomal cells along the rostro-caudal axis. This expression is transient and Myf-6 mRNA can no longer be detected in myotomal cells after day 12 post coitum (p.c.). In contrast to other muscle determination genes (MyoD1, myogenin, Myf-5), Myf-6 mRNA is not detected in limb buds or visceral arches and skeletal muscle of the mouse embryo (day 8-15 p.c.). In fetal mice, Myf-6 transcripts appear at day 16 p.c. in all skeletal muscles, and the gene continues to be expressed at a high level after birth. These results suggest that early Myf-6 expression may be restricted to a population of myogenic cells that does not contribute to the embryonic muscle masses in limb buds and visceral arches. The reappearance of Myf-6 mRNA in fetal skeletal muscle coincides approximately with secondary muscle fiber formation and the onset of innervation.

The muscle regulatory protein myogenin accumulates in differentiating muscle cells when the culture medium is depleted for serum. To investigate the regulation of myogenin gene expression, we have isolated and characterized the Myf4 gene which encodes the human homologue of murine myogenin. Serum components, basic FGF (b-FGF), transforming growth factor beta (TGF-beta), and EGF, agents which suppress differentiation of muscle cells in vitro, down-regulate the activity of the Myf4 gene, suggesting that it constitutes a nuclear target for the negative control exerted by these factors. The 5' upstream region containing the Myf4 promoter confers activity to a CAT reporter plasmid in C2C12 myotubes but not in fibroblasts and undifferentiated myoblasts. Unidirectional 5' deletions of the promoter sequence reveal that integral of 200 nucleotides upstream of the transcriptional start site are sufficient for cell type-specific expression. The forced expression of the muscle determining factors, MyoD1, Myf5, and Myf6 and to a lesser degree Myf4, results in the transactivation of the Myf4 promoter in C3H mouse 10T1/2 fibroblasts. Pathways potentially involved in conveying signals from the cell-surface receptors to the Myf4 gene were probed with pertussis- and cholera toxin, forskolin, and cAMP. Dibutyryl-cAMP and compounds that stimulate adenylate cyclase inhibit the endogenous Myf4 gene and the Myf4 promoter in CAT and LacZ reporter constructs. Conversely, pertussis toxin which modifies Gi protein stimulates Myf4 gene expression. In summary, our data provide evidence that the muscle-specific expression of the Myf4 gene is subject to negative control by serum components, growth factors and a cAMP-dependent intracellular mechanism. Positive control is exerted by a pertussis toxin-sensitive pathway that presumably involves G proteins.

A potent muscle-specific enhancer element, originally described in the rat myosin light chain (MLC) 1/3 locus located downstream of the coding region, is found in an analogous position in the human MLC1/3 gene. When linked to a CAT reporter gene and transfected into muscle or non-muscle cells, the human MLC enhancer directs high levels of muscle-specific gene expression from homologous or heterologous promoters, irrespective of position or orientation relative to the CAT transcription unit. A significant degree of sequence homology (over 85%) in the 3'-flanking regions of the two MLC genes is restricted to a 200 bp sequence which lies approximately 1.5 kb downstream of the polyadenylation site in both species. The human enhancer sequence includes binding sites for human myogenic determination factors containing a common basic helix-loop-helix motif, and it can be trans-activated to varying degrees in non-muscle cells by these factors. This study establishes the MLC enhancer as an evolutionarily conserved, integral component of the MLC1/3 locus which constitutes a novel target for the action of myogenic determination factors.

The Myf-6 gene, a novel member of the human gene family of muscle determination factors has been detected by its highly conserved sequence coding for a putative helix-loop-helix domain. This sequence motif is a common feature of all Myf factors and other regulatory proteins. The new Myf gene is located on human chromosome 12, approximately 6.5 Kb upstream of the Myf-5 locus in a closely linked cluster of myogenic determination genes. Myf-6 cDNAs were isolated from human and mouse skeletal muscle, the only tissue in which expression of the corresponding mRNA was observed. In contrast to human primary muscle cell cultures which express moderate levels of Myf-6 mRNA, most established rodent muscle cell lines completely lack this mRNA. Myogenic 10T1/2 cells, however, induced by the expression of either pEMSV-Myf-4 or pEMSV-Myf-5 activate their endogenous mouse Myf-6 gene. Constitutive expression of Myf-6 cDNA in C3H 10T1/2 fibroblasts establishes the muscle phenotype at a similar frequency to the previously characterized myogenic factors. Moreover, muscle-specific CAT reporter constructs containing either the human myosin light chain (MLC) enhancer or the promoter of the embryonic myosin light chain gene are activated in NIH 3T3 fibroblasts or in CV1 kidney cells by cotransfection of Myf-6 expression vehicles. This transcriptional activation occurs in the absence of any apparent conversion of the cellular phenotype of the recipient cells. Glutathione-S-transferase fusion proteins with Myf-3, Myf-4 or Myf-5 specifically bind to a MEF-like consensus sequence present in the human MLC enhancer and the MLC1 emb promoter. In contrast, the Myf-6 hybrid protein interacts weakly with the same sequences showing lower affinity and reduced specificity. Since co-expressed pEMSV-Myf-6, nevertheless, is able to activate transcription of the MLC-CAT reporter constructs in non-muscle tissue culture cells, the different DNA binding properties in vitro might suggest that transactivation of gene expression by Myf-6 involves distinct binding sites and/or additional protein factors.

The development of muscle cells involves the action of myogenic determination factors. In this report, we show that human skeletal muscle tissue contains, besides the previously described Myf-5, two additional factors Myf-3 and Myf-4 which represent the human homologues of the rodent proteins MyoD1 and myogenin. The genes encoding Myf-3, Myf-4 and Myf-5 are located on human chromosomes 11, 1, and 12 respectively. Constitutive expression of a single factor is sufficient to convert mouse C3H 10T1/2 fibroblasts to phenotypically normal muscle cells. The myogenic conversion of 10T1/2 fibroblasts results in the activation of the endogenous MyoD1 and Myf-4 (myogenin) genes. This observation suggests that the expression of Myf proteins leads to positive autoregulation of the members of the Myf gene family. Individual myogenic colonies derived from MCA C115 cells (10T1/2 fibroblast transformed by methylcholanthrene) express various levels of endogenous MyoD1 mRNA ranging from nearly zero to high levels. The Myf-5 gene was generally not activated in 10T1/2 derived myogenic cell lines but was expressed in some MCA myoblasts. In primary human muscle cells Myf-3 and Myf-4 mRNA but very little Myf-5 mRNA is expressed. In mouse C2 and P2 muscle cell lines MyoD1 is abundantly synthesized together with myogenin. In contrast, the rat muscle lines L8 and L6 and the mouse BC3H1 cells express primarily myogenin and low levels of Myf-5 but no MyoD1. Myf-4 (myogenin) mRNA is present in all muscle cell lines at the onset of differentiation.(ABSTRACT TRUNCATED AT 250 WORDS)

Three tRNAs specific for methionine, phenylalanine and tyrosine were isolated from the total tRNA of Bacillus subtilis by chromatographic procedures using BD-cellulose and reversed-phase (5) chromatography. The acceptor activities of the purified tRNAs are 1160, 1260 and 1320 pmoles per A260nm unit for tRNAMetf, tRNAPhe and tRNATyr2 respectively. In tRNAMetf and tRNAPhe ribothymidine, pseudouridine and dihydrouridine are present, in addition, in tRNAPhe 7-methylguanosine and a 2′-O-methylated nucleoside were found. The modified nucleosides of tRNATyr2 are ribothymidine, pseudouridine, dihydrouridine, 4-thiouridine and 1-methyladenosine. The results suggest the presence of 2-methylthio-N6-(Δ2-isopentenyl)adenosine in tRNAPhe and tRNATyr2. The thermal denaturation profiles of the three tRNA species are presented.

The combination of DNA microinjection into tissue culture cells and the chloramphenicol acetyltransferase enzyme assay has been used to determine quantitatively the transcriptional activity of viral or eukaryotic promoters. As shown for a wide range of DNA concentrations and time periods between 2 and 16 h the assay allows the reliable and reproducible determination of promoter activities in defined cellular backgrounds. Only 50 cells are necessary for the test.

A 7-methylguanine (m7G) specific tRNA methyltransferase from E. coli MRE 600 was purified about 1000 fold by affinity chromatography on Sepharose bound with normal E. coli tRNA. The purified enzyme catalyzes exclusively the formation of m7G in submethylated bulk tRNA of E. coli K12 met- rel-. The purified enzyme transfers the methyl group from S-adenosyl-methionine to initiator tRNA of B. subtilis and 0.8 moles m7G residues are formed per mole tRNA. It is suggested that the enzyme specifically recognizes the extra arm unpaired guanylate residue.

The chicken cytoplasmic beta actin gene is ubiquitously expressed in all cell types. In terminally differentiated muscle cells, however, the concentration of beta actin specific mRNA is down-regulated to scarcely detectable levels. To test for gene regions which are involved in the muscle specific reduction of beta actin specific mRNA, the isolated complete chicken beta actin gene or chimeric gene constructs containing parts of the gene were stably transfected into the myogenic mouse cell line C2C12 and their transcriptional activity was compared in proliferating myoblasts and postmitotic myotubes. A hybrid construct containing the beta actin promoter fused to the bacterial CAT gene showed high and constitutive expression during myocyte differentiation. In contrast, constructs containing the SV40 early promoter linked to the 3' end of the beta actin gene led to a marked reduction of beta actin transcripts in differentiated C2C12 myotubes. The stability of beta actin mRNA was analyzed in actinomycin D treated cells and found to be virtually unchanged in myotubes as compared to myoblasts. These results suggest that a sequence element located in the 3' end or 3' flanking region of the beta actin gene confers the myotube specific down-regulation that is not primarily due to destabilization of mRNA.

The major methylated bases of tRNA of Bacillus subtilis are 5-methyluracil (m5U), 7-methylguanine (m7G) and 1-methyladenine (m1A). In addition, tRNA from stationary phase cultures always contains considerable amounts of an unidentified methylated pyrimidine derivative, “P.” Trimethoprim, which inhibits tetrahydrofolate (FH4)-dependent transmethylation reactions, was used to accumulate m5U-deficient tRNA in B. subtilis. With Escherichia coli enzymes the methyl moiety of S-adenosylmethionine (SAM) can be incorporated into the tRNA, and 0.7 m5U residue is formed per one tRNA molecule. The FH4-dependent formation of m5U in tRNA of B. subtilis as well as SAM-dependent methylations are affected by antibiotics which interfere with ribosomal functions. tRNA from pactamycin- or chloramphenicol-treated cells serves as substrate for SAM-dependent m5U- and m7G-specific tRNA methyltransferases from E. coli. With homologous SAM-dependent tRNA methyltransferases considerably fewer, but specific, transmethylations were found. tRNA obtained from a methionine-requiring stringent mutant of B. subtilis starved for methionine in the presence of chloramphenicol accepts the methyl moiety from SAM with homologous enzymes. The pattern of the methylated bases upon in vitro methylation of this tRNA agrees well with the pattern of methioninederived methylated bases in total tRNA of B. subtilis. Significant alterations were found in the relative distribution of tRNA isoacceptors from pactamycin-treated B. subtilis, which may reflect an accumulation of premature tRNAs.

Human smooth muscle and nonmuscle cells express closely related myosin alkali light chains which are different from the isoforms present in striated muscle tissues. To dat no information of the amino acid sequence of these mammalian nonstriated muscle isoforms has been available. We have isolated full-length cDNA clones encoding the nonmuscle and smooth muscle myosin light chains from cultured human lymphoblasts and heart aorta smooth muscle cells, respectively. Here we present the complete nucleotide sequences for both cDNA clones, together with the deduced amino acid sequences for the peptides. Both cDNAs contain the same open reading frame for 151 amino acids with 5 amino acid differences located in the C terminus. These differences are encoded by a block of 44 nucleotides with is present only the smooth muscle mRNA. To identify the human gene coding for the two MLC isoforms, we have isolated and sequenced the nonmuscle MLC gene, together with several intronless pseudogenes. A singl e functional gene was found containing 7 exons which are utilized for the coding information of the SM MLC mRNA. In contrast, the NM MLC mRNA does not contain sequences encoded by exon 6 which corresponds to the 44 nucleotides sequences encoded by exon 6 which corresponds to the 44 nucleotides expressed in SM mRNA. This genomic configuration suggests that both the smooth muscle and nonmuscle MLCs in man are generated from the identical primary transcript by alternative splicing pathways taking place in a tissue-dependent manner.

A set of cDNA clones coding for alkali myosin light chains (AMLC) was isolated from fetal human skeletal muscle. Nucleotide sequence analysis and RNA expression patterns of individual clones revealed related sequences corresponding to (i) fast fiber type MLC ! and MLC 3 ; (ii) the embryonic MLC that is also expressed in fetal ventricle and adult atrium (MLC emb ); and (iii) a nonsarcomeric MLC isoform that is found in all nonmuscle cell types and smooth muscle. The AMLC gene family in man comprises unique copies for MLC 1 , MLC 3 and MLC emb , and multiple copies for the nonsarcomeric MLC genes. The gene coding for MLC 1 and MLC 3 is located on human chromosome 2.

Transcriptional regulation of the chicken cardiac myosin light chain 2 (MLC2-A) gene was investigated in chicken primary myoblast and fibroblast cultures transfected with vector constructs containing the bacterial marker gene for chloramphenicol acetyltransferase (CAT) under the control of the MLC2-A promoter. We here demonstrate that sequences close to the TATA box are sufficient to direct muscle specific and regulated expression of the MLC2-A mRNA. Transcription from MLC2-A promoter/CAT hybrids in myocytes starts from the authentic cap site that is also used in vivo. In primary breast muscle cells, bromodeoxyuridine (BUdR), a reversible blocking agent of cell differentiation, suppresses transcription from the MLC2-A promoter whereas nonmuscle promoters like the RSV- or the cytoplasmic beta-actin promoter are unaffected in their transcriptional capacity. Although the endogenous cardiac MLC2-A gene in chicken is exclusively active in heart, the transfected MLC 2-A promoter escapes this cell type control in primary cultures of breast muscle. These results demonstrate that although muscle specificity of the MLC2-A gene and its transcriptional up-regulation during differentiation is maintained in a rather short promoter segment, restrictive elements determining the muscle cell type specificity in vivo are either not present in our constructs or are not acting under the conditions of transient transfection.

In mammalian organisms the regulatory or phosphorylatable myosin light chains in heart and slow skeletal muscle have been shown to be identical and presumably constitute the product of a single gene. We analyzed the expression of the avian cardiac myosin light chain (MLC) 2-A in heart and slow skeletal muscle by a combination of experimental approaches, e.g., two-dimensional gel electrophoresis of the protein and hybridization of mRNA to specific MLC 2-A sequences cloned from chicken. The investigations have indicated that, unlike in mammals, in avian organisms the phosphorylatable myosin light chains from heart and slow skeletal muscle are distinct proteins and therefore products of different genes. The expression of MLC 2-A is restricted to the myocardium and no evidence was found that it is shared with slow skeletal muscle.

The nucleotide sequence of a cDNA clone (pML10) for chicken cardiac myosin light chain is described. The cDNA insert contains 613 nucleotides representing the entire coding sequence, with the exception of nine NH2-terminal amino acids, and the full 3'-non-coding region of 146 nucleotides. The missing 5' terminus of the mRNA, not represented in the clone pML10, was obtained by extension of the cDNA using a 43 nucleotide long internal EcoR1 fragment as a primer. The non-coding region contains several direct and inverted repeated sequences and the polyadenylation signal sequence AATAAA. The coding portion exhibits non-random usage of synonymous codons with a strong bias for codons ending in G and C.

We have isolated cDNA recombinant phages encoding the embryonic isoform of the myosin alkali light chain (MLC1emb) from a human fetal skeletal muscle library. The cDNA clones were detected by their weak cross-hybridization to a human MLC1F and MLC3F cDNA clone. Nucleotide sequence analysis of the complete cDNA (GT14) revealed an open reading frame for 197 amino acids. The derived protein sequence constitutes the first structural information on this myosin isoform of any organism. Remarkable structural similarities to other alkali MLC polypeptides, particularly to those of the slow-muscle type, are evident. Under conditions of high stringency, the GT14 clone hybridized to an abundant mRNA species in fetal ventricular muscle and adult atrial muscle, whereas in fetal skeletal muscle only a very weakly hybridizing mRNA component was detected. These mRNAs were indistinguishable by size and the thermal stability of their hybrids formed with the DNA insert of clone GT14. We therefore conclude that identical mRNA is expressed in these tissues, presumably transcribed from the same gene. According to its pattern of mRNA expression, the novel MLC isoform described here was designated as “embryonic and atrial myosin light chain” (MLC1emb/A) in reference to its developmental stage-specific and tissue-specific appearance in embryonic skeletal muscle, fetal ventricle and adult atrium.

tRNA I Tyr and tRNA II Tyr have been purified from B.subtilis and their nucleotide sequence determined. tRNA I Tyr differs from tRNA II Tyr only by the extent of modification of the adenosine in 3′ position adjacent to the anticodon, i 6 A and ms 2 i 6 A respectively.

We have isolated the cDNA encoding a novel human myogenic factor, Myf-5, by weak cross-hydridization to the mouse MyoD1 probe. Nucleotide sequence analysis and the identification of the corresponding gene indicate that human Myf-5 is a member of a small gene family which also contains the human homologue to MyoD1. Although structurally related to the mouse factor, the human Myf-5 constitutes a different protein which nevertheless is capable of inducing the myogenic phenotype in embryonic C3H mouse 10T1/2 'fibroblasts'. The existence of more than one MyoD1-like protein in human skeletal muscle is further suggested by the detection of several similar but distinct cDNA clones. The phenotypic conversion of 10T1/2 cells by the human factor is recognized by the capacity of the cells to form multinucleated syncytia and synthesize sarcomeric myosin heavy chains. Moreover, transient expression of Myf-5 in 10T1/2 cells leads to the activation of a co-transfected muscle-specific CAT reporter gene which by itself is transcriptionally silent in the non-muscle cell background. The deduced amino acid sequence of clone Myf-5 reveals a region which is highly similar to myc proteins and the developmental factors from Drosophila encoded by the achaete scute locus and the twist gene. The myc homology region and a preceding cluster of basic amino acids are located in a larger sequence domain with strong similarity to the mouse myogenic factor MyoD1. Two additional short segments with high serine and threonine content are conserved between the two proteins.(ABSTRACT TRUNCATED AT 250 WORDS)